NLRP3-Inflammasome Inhibition with IZD334 Does Not Reduce Cardiac Damage in a Pig Model of Myocardial Infarction

Figure A1. Regional cardiac function and dobutamine echocardiography in animals treated with placebo or 10 mg/kg IZD334. (A) Septal and inferolateral systolic wall thickening; SWT (%) at baseline and 7 days after MI with or without dobutamine. (B) Systolic wall thickness; WT (cm) (n = 5–7).

Figure A2. Histological analysis of infarct tissue (n = 6–7). (A) Collagen formation. (B) Immune cell infiltration.

Figure A3. Neutrophil (A) and monocyte (B) infiltration (n = 6–7).

Figure 1. In vitro reduction of IL-1β secretion. (A) Porcine peripheral blood mononuclear cells (PBMCs) released IL-1β after administration of lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in vitro. Addition of IZD334 reduced IL-1β release (n = 3). (B) Whole blood IC50 calculation after stimulation with LPS and ATP and administration of increasing concentrations of IZD334 in vitro. p-values (** <0.005, *** <0.0005) are compared to vehicle control (0 µM of IZD334).

Figure 2. Ex vivo stimulation assay after in vivo IZD334 infusion. IL-1β release was significantly reduced when porcine whole blood was stimulated ex vivo after infusion of IZD334.

Figure 3. Plasma concentrations of IZD334 in healthy pigs. Concentrations following intravenous (IV) and oral (PO) administration.

Figure 4. Serological and histological inflammatory read-outs. (A) Circulating levels of leukocytes and (B) neutrophils during the 7-day follow-up period. (C) Circulating levels of C-reactive protein (CRP) during the 7-day follow-up. (D) Myocardial interleukin (IL)-1β levels, measured in infarct (I) and border zone (BZ) regions of the pig heart. Data are presented as mean ± SEM (p-values following mixed models testing). N.B. The first three timepoints of the analysis are baseline, 4 h after reperfusion, and 24 h.

Figure 5. Representative pictures of basal slices of the different treatment groups (placebo, 1 mg/kg IZD334, 3 mg/kg IZD334, and 10 mg/kg IZD334. The dark area represents the remote area, the AAR is stained red, and the infarcted myocardium is stained white/pale.

Figure 6. Myocardial infarct size. Normal distribution of data was confirmed as described in our methods (using Shapiro–Wilk test). Hence, a one way-ANOVA was performed. (A) The area at risk as a ratio of the left ventricle (AAR/LV) was similar in all treatment groups (placebo 26.8 ± 6.1%, 1 mg/kg IZD334 23.5 ± 3.2%, 3 mg/kg IZD334 26.0 ± 1.9%, 10 mg/kg IZD334 26.4 ± 6.6%, ANOVA, p = 0.67) groups (p = 0.67). (B) The infarct size (IS) was not different between treatment groups when expressed relative to measurements of the ischemic AAR (placebo 73.1 ± 3.0%, 1 mg/kg IZD334 75.5% ± 7.3%, 3 mg/kg IZD334 80.3 ± 3.9%, 10 mg/kg IZD334 78.2% ± 8.0%, ANOVA p = 0.21). (C) IS as percentage of the LV did not differ (placebo 19.6 ± 4.5%, 1 mg/kg IZD334 17.7 ± 6.3%, 3 mg/kg IZD334 20.5 ± 2.2%, 10 mg/kg IZD334 19.3 ± 2.9%, ANOVA p = 0.58). N.B. In one pig of the 3 mg/kg group, it was not possible to measure the AAR due to a failure to infuse Evans Blue in the coronary arteries. Therefore, only in 6 pigs could the AAR and the IS/AAR be shown.

Figure 7. Cardiac troponin levels. Circulating levels of troponin at baseline and after reperfusion (4 h, 24 h, and prior to termination). Data are presented as mean ± SEM (p-values following mixed models testing).

Figure 8. Global cardiac function measured by cardiac magnetic resonance imaging. Normal distribution of data was confirmed as described in our methods (using Shapiro–Wilk test). Hence, a one way-ANOVA was performed. No differences were observed among groups. (A) End diastolic volume (EDV) (placebo 140 ± 21 mL, 1 mg/kg IZD334 150 ± 26 mL, 3 mg/kg IZD334 151 ± 8 mL, 10 mg/kg IZD334 144 ± 22 mL, ANOVA p = 0.78). (B) End systolic volume (ESV) placebo 81 ± 20 mL, 1 mg/kg 72 ± 19 mL, 3 mg/kg 87 ± 8 mL, 10 mg/kg 80 ± 19 mL, ANOVA p = 0.49. (C) Ejection fraction percent (EF%) (placebo 45.1 ± 8.7%, 1 mg/kg 49.9 ± 6.1%, 3 mg/kg 42.7 ± 3.8%, 10 mg/kg 44.9 ± 6.4%, ANOVA p = 0.26).

Figure 9. In vivo IZD334 plasma concentration measurements. Plasma measurements of IZD334 levels revealed peak concentrations 4 h (h) post-IR in all groups, consistent with an initial dosing of 10 mg/kg. Plasma levels during follow-up were consistent within each group and increased with dose across the dose range (1, 3, and 10 mg/kg IZD334).